Project Summary The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. In order to prevent patients from prematurely interrupting their treatment, potentially resulting in the emergence of drug-resistant TB, DOTS (Directly Observed Treatment, Short-course) programs have been set-up where patients are continuously supervised during the full length of the treatment to ensure that they do take their medicine. Shortening the duration of the treatment could significantly prevent mismanagement of TB chemotherapy and development of drug resistance. We have discovered that small thiols such as cysteine, dithiothreitol or N- acetylcysteine (NAC), sterilize cultures of Mycobacterium tuberculosis (Mtb) with no emergence of drug-resistant mutants when combined with the first line TB drugs isoniazid (INH) or rifampicin (RIF). The mechanism of sterilization is based on the ability of these thiols to 1) induce an iron-dependent oxidative stress and 2) reduce menaquinone into menaquinol, the driver of Mtb respiration. The coercion of Mtb into consuming oxygen keeps the Mtb cells in a constant active metabolic state preventing the formation of persisters, a small fraction of Mtb cells refractory to drugs and immune action, and allowing the TB drugs to stay bactericidal. The combination of NAC and INH was tested in Mtb-infected murine macrophages and found to potentiate the killing effect of INH. Based on these preliminary experiments, we propose to determine whether 1) NAC could boost the killing activity of other first-line, and second-line TB drugs in vitro and in macrophage models against drug-susceptible and drug-resistant Mtb; 2) NAC prevents the formation of persisters in drug-treated Mtb; 3) iron is required for NAC activity in macrophages; and 4) NAC combined with TB drugs potentiates drug killing of drug- susceptible and drug-resistant Mtb strains in murine models of Mtb infection. This is an innovative proposal with high risks and high potential rewards. If successful, this proposal will impact the worldwide TB community providing an adjunct therapy with NAC which is already used to treat drug toxicity in TB patients. NAC could therefore be easily added to the TB pharmacopeia to substantially improve chemotherapy outcome and to prevent emergence of drug resistance.